Molecular characterization of HIV-1 strains collected from around the world has revealed extensive genetic diversity. Based on phylogenetic analysis of viral genomic sequences, HIV-1 has been divided into three distinct groups, M, N and O. Group M viruses represent the majority of HIV-1 and based on sequence divergence have been further subdivided into nine distinguishable clades, designated subtypes A, B, C, D, F, G, H, J, and K (Robertson, D. L. et.al. In: Human Retroviruses and AIDS 1999-A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences, Kuiken, C. et. al. Eds., pgs. 492-505 (1999)). The phylogenetic pattern for group M isolates has been described as a star phylogeny with the subtypes roughly equidistant from each other while diverging from a common ancestor. For viral envelope (env) gene amino acid sequences, the degree of intrasubtype divergence ranges up to 20% and the intersubtype divergence is 25-30% (Sharp, P. M. et.al., AIDS 8: S27-S42 (1994)).
In 1990, an unusual HIV-1 strain (ANT70) isolated from a Cameroonian patient was reported (De Leys, R. et. al., J. Virol. 64:1207-1216 (1990)). Based on the available sequence information, this strain of virus appeared to be very different from other HIV-1 sequences. A similar virus (MVP-5180) was isolated from a second Cameroonian patient (Gürtler, L. et. al., J. Virol. 68:1581-1585 (1994)). Complete genome sequencing revealed that although these viruses shared the same overall genomic structure with group M strains, their sequences were highly divergent having only ˜50% nucleotide homology within the env gene as compared to group M isolates (Gürtler, L. et. al., J. Virol. 68:1581-1585 (1994)). Due to the extent of genetic divergence from group M strains, these isolates were designated as group O (outlier) viruses. More recently, HIV-1 viruses that are phylogenetically equidistant from group M and group O strains have been identified in Cameroon; these have been designated as group N (Simon, F. et. al., Nat. Med. 4:1032-1037 (1998)).
An innately error-prone reverse transcriptase enzyme, high viral loads and in vivo selective pressure all contribute to the genetic diversity of HIV-1. An additional source of diversity is a by-product of the HIV replicative cycle where two genomic RNA transcripts linked at their 5′ ends are encapsidated into a virion. If a cell is simultaneously infected with more than one HIV-1 strain, heterozygous virions can be produced. Subsequent to infection with the virion, reverse transcriptase can switch back and forth between the two RNA transcripts, generating a recombinant virus (Hu, W. S. and H. M. Temin, Science 250:1227-1233 (1990)). This capacity to recombine provides an opportunity for rapid and dramatic genetic change. A naturally-occurring intersubtype recombinant virus was first identified by Sabino and colleagues who characterized a B/F mosaic found in two epidemiologically linked patients (Sabino, E. C. et. al., J. Virol. 68:6340-6346 (1994)). In areas where multiple subtypes co-circulate, intersubtype recombinants may account for 20% or more of HIV-1 infections (Cornelissen, M. et. al., J. Virol. 70:8209-8212 (1996)). Although the majority of viral recombinants described to date are group M intersubtype mosaics, intergroup recombinant viruses composed of group M and group O gene segments have also been identified (Peeters, M. et. al., J. of Virol. 73:7368-7375 (1999)).
Characterization of full-length genomes revealed that reference strains for two previously recognized subtypes of group M were actually intersubtype recombinant viruses. All representatives of “subtype E” strains sequenced to date consist of gag and RNA dependent DNA polymerase (pol) genes from subtype A while their env gene is derived from subtype E (Gao, F. et. al., J. Virol. 70:7013-7029 (1996)). HIV-1 strains previously recognized as subtype I strains have since been shown to be triple mosaics consisting of subgenomic segments derived from subtypes A, G and I (Nasioulas, G. et. al., AIDS Res. Hum. Retroviruses 15:745-758 (1999)). Such recombinant strains with evidence of epidemic spread have been classified as Circulating Recombinant Forms (CRF; Robertson, D. L. et.al. In: Human Retroviruses and AIDS 1999-A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences, Kuiken, C. et. al. Eds., pgs. 492-505 (1999)).
The potential for emergence of CRF strains is well documented. Subtype E strains, designated CRF01_AE, are the predominant form of HIV-1 in Thailand. In Kaliningrad, an outbreak of an A/B recombinant virus (CRF03_AB) has recently been documented in injecting drug users (Liitsola, K. et. al., AIDS 12:1907-1919 (1999)). An A/G intersubtype recombinant with a unique and complex mosaic pattern (CRF02_AG), has been identified in Nigeria, Djibouti and regions of west central Africa (Carr, J. K. et. al., Virology 247:22-31 (1998)).
The overall distribution of HIV-1 groups, subtypes and CRFs varies considerably in different geographic regions and is undergoing continual change. While subtype B is predominant in North America and Western Europe (McCutchan, F. E., AIDS 14 (suppl 3): S31-S44 (2000)), increasing numbers of non-subtype B infections are being observed in both Europe and the United States. In France, over the 10-year period from 1985-1995, the prevalence of non-B viruses increased from approximately 4% to more than 20% (Barin, F. et. al., AIDS 11:1503-1508 (1997)). Non-B reactive specimens were found in almost all regions tested. Remarkably, nearly every group M subtype and group O infections were reported at a single hospital in Paris (Simon, F. et. al., AIDS Res. Hum. Retroviruses 15:1427-1433 (1996)). Analysis of 24 recently infected German patients revealed that 33% were infected with non-B viruses; these included subtypes A, E and C (Dietrich, U. et. al., AIDS 11:1532-1533 (1997)). In Belgium, subtype A, C, D, E, F, G and H infections were detected, accounting for more than 30% of total HIV-1 infections (Heyndrickx, L. et. al., AIDS Res. Hum. Retroviruses 14:1291-1296 (1998)). Increasing numbers of non-subtype B infections, including subtypes A, D, E, F and group O, are also being detected in the United States (Weidle, P. J. et. al., J. Infect. Dis. 181:470-475 (2000). Thus, viral heterogeneity is increasing in regions in which subtype B was traditionally most prevalent.
Quantification of virion-associated RNA in plasma has become a well-established method for clinical management and follow-up of patients with HIV-1 infection. A variety of nucleic acid-based techniques have been developed for detection and quantification of HIV-1 viral RNA including, reverse transcriptase-coupled polymerase chain reaction (RT-PCR), nucleic acid sequence-based amplification (NASBA), and branched DNA (bDNA) (Mulder, J. et. al., J. Clin. Microbiol. 32:292-300 (1994); Kievits, T. et. al., J. Virol. Methods 35:273-286 (1991); Kern, D. et. al., J. Clin. Microbiol. 34:3196-3202 (1996); Swanson P. et. al., J. Virol. Methods 89:97-108 (2000)). These techniques all rely on hybridization of oligonucleotides to the target sequences. Mismatches between the primers/probes and target sequences have the potential to abolish or reduce the efficiency of amplification and/or detection of the targeted sequences. Thus, selection of primer and/or probe sequences plays a critical role in the performance of these assays.
The original nucleic acid-based tests were developed based primarily on sequence information derived from HIV-1 subtype B common to the United States and Western Europe. The influence of HIV-1 genetic diversity on the efficiency of amplification by the first-generation Amplicor HIV-1 Monitor (version 1.0) assay soon became evident as it failed to detect or underquantified group M subtype A, E, F, G and group O clinical specimens and viral isolates (Loussert-Ajaka, I. et. al., Lancet 346:912-913 (1995); Coste, J. et. al., J. Med. Virol. 50:293-302 (1996); Swanson P. et. al. J. Virol. Methods 89:97-108 (2000)). Mismatches due to HIV-1 genetic diversity were also shown to affect quantification of group M subtype A, G, H, J, and group O specimens by the NASBA HIV-1 RNA QT test (Coste, J. et. al., J. Med. Virol. 50:293-302 (1996); Vandamme, A-M. et. al., J. Acquired Immune Defic. Syndr. Hum. Retrovirol. 13:127-139 (1996); Debyser, Z. et. al., AIDS Res. Hum. Retroviruses 14:453-459 (1998)). Intrasubtype diversity also impacts these assays as both the Amplicor HIV-1 Monitor and the NASBA HIV RNA QT test underquantified genetically divergent subtype B specimens (Alaeus, A. et. al., AIDS 11:859-865 (1997); Gobbers, E. et. al., J. Virol. Methods 66:293-301 (1997)). The influence of HIV-1 genetic diversity on assay performance was still evident even on second-generation versions of the RT-PCR, NASBA and bDNA assays (Segondy, M. et. al., J. Clin. Microbiol. 36:3372-3374 (1997); Holguin A., et. al., Eur. J. Clin. Microbiol. Infect. Dis. 18:256-259 (1999)). The current Amplicor Monitor 1.5 test shows marked improvement on group M subtypes, but fails to detect or quantifies unreliably, group O specimens (Swanson P. et. al., J. Virol. Methods 89:97-108 (2000)). The gag-based NASBA and bDNA assays also fail to detect or underquantify group O specimens (Gobbers, E. et. al., J. Virol. Methods 66:293-301 (1997), Swanson P. et. al., J. Clin. Micro. 39:862-870 (2001)).
Due to the ever-changing geographical distribution of HIV-1 groups and subtypes and the increasing numbers of recombinant forms of HIV-1, it has become critical that assays used to monitor HIV-1 RNA levels in plasma be capable of detecting all HIV-1 variants. Ideally, assays used to quantify HIV-1 viral RNA should function in a group- and subtype-independent manner to ensure reliable quantification of all infections.
Further compounding the difficulty in finding a primer set capable of initially hybridizing with the various groups and subtypes of the highly mutable HIV-1 genome, is the fact that primers selected by comparing them to various genomes are not necessarily effective for amplifying the intended target. As described in He Q., et al., BioTechniques, Vol. 17, No. 1, pp 82-86 (1994), those skilled in the art experience unexplained difficulties obtaining a significant amplification product from primer sets that hybridize to a selected target sequence. This yet to be explained phenomenon has been a challenge facing those designing primer sets for a given target sequence and further complicates the choice of primers for an already difficult HIV-1 target.
There is therefore a need for primer sets and reagents for specifically and sensitively amplifying and detecting HIV-1 variants including those from HIV-1 groups M, N, and O, as well as the various subtypes within or derived from these groups.